THE OBJECTIVE OF THIS RESEARCH PLAN IS TO ISOLATE A CDNA SEQUENCE ENCODING TUMOR NECROSIS FACTOR (TNF) AND, BY MANIPULATION OF THAT SEQUENCE, TO PRODUCE AND SECRETE THE MATURE TNF PROTEIN FROM THE YEAST SACCHAROMYCES CEREVISIAE.

THE OBJECTIVE OF THIS RESEARCH PLAN IS TO ISOLATE A CDNA SEQUENCE ENCODING TUMOR NECROSIS FACTOR (TNF) AND, BY MANIPULATION OF THAT SEQUENCE, TO PRODUCE AND SECRETE THE MATURE TNF PROTEIN FROM THE YEAST SACCHAROMYCES CEREVISIAE. PREVIOUS WORK ON TNF HAS SHOWN IT TO HAVE CYTOSTATIC OR CYTOLYTIC ACTIVITY IN VITRO AGAINST A NUMBER OF TRANSFORMED CELL LINES AND TO HAVE TUMOR NECROTIC ACTIVITY AGAINST VARIOUS TUMORS IN ANIMAL MODELS. MOST IMPORTANTLY, IT HAS NO ADVERSE EFFECT ON NORMAL CELLS EITHER IN CULTURE OR IN ANIMAL MODELS. THIS SPECIFICITY FOR TUMOR CELLS SUGGESTS A POTENTIAL VALUABLE THERAPEUTIC ROLE FOR TNF IN TREATING HUMAN TUMORS. IN ORDER TO ESTABLISH ITS VALUE, HOWEVER, SUFFICIENT QUANTITIES OF THIS RATHER RARE PROTEIN MUST BE ISOLATED FOR TESTING. THE OVERALL AIM OF THIS RESEARCH PROPOSAL IS TO PRODUCE TNF ECONOMICALLY IN LARGE QUANTITIES FROM YEAST CELLS. AS A RESULT OF THE PHASE I EFFORTS DESCRIBED HERE, A CDNA COPY OF THE TNF GENE WILL BE ISOLATED AND CHARACTERIZED. IN PHASE II, THE PORTION OF THE GENE CODING FOR MATURE TNF WILL BE ATTACHED TO YEAST PROMOTER AND SECRETION SIGNAL SEQUENCES, AND THE ENTIRE TRANSCRIPTIONAL UNIT WILL BE INTEGRATED STABLY INTO THE YEAST GENOME. THE RESULT OF PHASE II SHOULD BE A GENETICALLY STABLE YEAST STRAIN SECRETING 1020 MG OF TNF PER LITER OF MEDIUM.